Study Protein Denaturation Research Articles

Optical techniques to determine thermal effects on proteins

SummaryOptical rotation (OR) and transmitted light (TL) measurements were conducted on 1%, 2.5% and 5% (w/v) bovine serum albumin (BSA) in 0.01 m phosphate buffer at pH 7 and ionic strength 0.08. Denaturation temperatures (Td) obtained from OR measurements were consistent with reported differential scanning calorimetry values. Protein concentration did not affect Td in agreement with most reports. Changes in TL reflecting gel formation and protein aggregation were influenced by BSA concentration. Sugar concentration in the range used in this study (0–5%) did not affect the thermal stability of BSA. The lack of difference in sucrose, trehalose and sorbitol effects on the thermal stability of BSA was consistent with some but not all reports. The optical system used to study protein denaturation had acceptable accuracy (consistency with published Td values) and precision (coefficient of variation under 3.5%) levels.

On the application of CZE to the study of protein denaturation

Experimental mobilities obtained from CZE are used to study protein denaturation through a model based on known physicochemical theories. This model is able to provide additional information concerning the folded and unfolded protein states from mobility data. Its use comprises first the evaluation of relevant parameters of the protein microstates like the electrostatic free energy, apart from the classical conformational free energy, and second the expression of raw experimental data concerning the folding-unfolding transition into more specific physicochemical parameters like protein hydrodynamic radius, net charge number, and hydration. Spurious effects that are intrinsic to the experimental evaluation of the mobility of protein states, like BGE viscosity, pH, and ionic strength variations accompanying the changes of the denaturant agent intensity are eliminated. In order to illustrate the proposal of this work, two case studies are considered here. The first one concerns thermal and urea denaturations of horse heart ferricytochrome c and the second one involves thermal denaturation of hen egg-white lysozyme. Thus, relevant theoretical thermodynamic considerations of the folded-unfolded protein transition are presented, where the electrostatic free energy is included explicitly in the effective free energy. It is found that this transition involves sharp increases of hydrodynamic radius and protein hydration.

Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry

Many methods exist for measuring and studying protein denaturation in vitro. However, measuring protein denaturation in cells under conditions relevant to heat shock presents problems due to cellular complexity and high levels of light scattering that interfere with optical techniques. A general method for measuring protein denaturation in cells using high sensitivity differential scanning calorimetry (DSC) is given. Profiles of specific heat ( c p vs. temperature) are obtained providing information about transitions in cellular components including the denaturation of proteins. The specific approaches employed with erythrocytes, bacteria, and mammalian cells are described, and an identification of several features of the DSC profiles is given. Protein denaturation on the level of roughly 7–20% occurs for commonly used heat shocks in mammalian cells.

In Situ Thermal Denaturation of Proteins in Dunning AT-1 Prostate Cancer Cells: Implication for Hyperthermic Cell Injury

The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure (from alpha helix to extended beta sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (approximately 41 degrees C at 2 degrees C min(-1) and approximately 44 degrees C at 5 degrees C min(-1)). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from approximately 110 kJ mol(-1) between 44 and 90 degrees C to approximately 750 kJ mol(-1) between 44 and 50 degrees C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between approximately 62 and 65 degrees C, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at the macromolecular level.

Computer Simulation of Urea−Water Mixtures: A Test of Force Field Parameters for Use in Biomolecular Simulation

A molecular model for urea to be used in conjunction with the simple point charge (SPC) model for liquid water in protein denaturation studies is validated by comparison of molecular dynamics (MD) simulation results to experimental data at 298 K as a function of urea mole fraction. The density, enthalpy of mixing, free enthalpy of urea hydration, and urea diffusion show very good agreement with the experimental values. The experimental error in the free enthalpy of hydration, which is dominated by the inaccuracy of the vapor pressure of solid urea, is larger than the simulation error. This limited accuracy does not allow a check of the nonideality of the solution. Free enthalpies have been obtained by thermodynamic integration. The importance of a correct use of the combinatorial factor in the partition function when interpreting simulation results obtained by thermodynamic integration is discussed. The tested, GROMOS96 compatible, force field parameters form a good basis for biomolecular simulations using urea−water mixtures.

Determination, by dynamic surface-tension analysis, of the molar mass of proteins denatured in guanidine thiocyanate.

A drop-based dynamic surface-tension detector (DSTD) has been used to study the dynamic surface tension behavior of proteins denatured in guanidine thiocyanate (GndSCN). The dynamic surface tension at the air-liquid interface is obtained by measuring the internal pressure of drops that grow and detach at a specified rate. In the method the sample of interest is injected and subsequently flows to the DSTD-sensing capillary tip. For this work, a novel DSTD calibration procedure utilizing two distinct mobile phases is applied. Here, the mobile phases are aqueous with different constituents, for example GndSCN and phosphate buffer, either added or omitted. The dual-mobile phase calibration procedure gives the analyst the capability of making protein measurements in a GndSCN-phosphate buffer mobile phase, while measuring a calibration standard in another mobile phase, such as water, in which the surface tension of the calibration standard is readily available. Results are presented with drop volumes of either 2 microL (i.e. 2-s drops) or 7 microL (i.e. 7-s drops) for proteins varying in molar mass from 12,000 to 330,000 g mol(-1). We demonstrate that the DSTD can be used to determine the molar mass of proteins denatured in GndSCN. The method applies a regime where the denatured protein is detected by surface-active properties, and selectivity with regard to molar mass is contained in the dynamic component of the DSTD signal. The dynamic surface pressure signals of the denatured proteins suggest that diffusion plays a large role in the kinetics of the surface activity. The limit of detection for the denatured proteins studied ranged from 3 mg L(-1) to 14 mg L(-1). The DSTD, coupled with the novel dual-mobile phase calibration procedure, can be used to investigate the fundamental properties of proteins. Insight into the behavior at the air-liquid interface for native and denatured proteins is achieved; this is a novel tool for studying protein denaturation, complementary to other common approaches such as spectroscopy and calorimetry. Furthermore, the reported method could be widely applied to the study of effects on the interfacial properties of proteins after a variety of chemical and physical modifications that are possible with the dual-mobile phase calibration procedure.

NMR studies of structure and function of biological macromolecules.

Nuclear magnetic resonance (NMR) spectroscopy is unique among the methods available for three-dimensional structure determination of proteins and nucleic acids at atomic resolution, since the NMR data can be recorded in solution. Considering that body fluids such as blood, stomach liquid and saliva are protein solutions where these molecules perform their physiological functions, knowledge of the molecular structures in solution is highly relevant. In the NMR experiments, solution conditions such as the temperature, pH, and salt concentration can be adjusted so as to closely mimic a given physiological fluid. Conversely, the solutions may also be changed to quite extreme non-physiological conditions, for example, for studies of protein denaturation. Furthermore, in addition to protein structure determination, NMR applications include investigations of dynamic features of the molecular structures, as well as studies of structural, thermodynamic and kinetic aspects of interactions between proteins and other solution components, which may either be other macromolecules or low molecular weight ligands. Again, for these supplementary data it is of keen interest that they can be measured directly in solution. The NMR structure of the Antennapedia homeodomain (Fig. 1) illustrates one of the exciting features of being able to perform structural studies in isolation. The polypeptide chain in this protein is only partially folded, with both chain ends showing pronounced disorder [1]. In the complex with its operator DNA (Fig. 2) the Nterminal chain end is located in the minor groove of the DNA, where it adopts a well-defined structure [2]. This mode of intermolecular recognition illustrates the functional importance of partially structured polypeptide chains. Mammalian prion proteins are an even more striking example of partial polypeptide folding, since the three-dimensional structure of the benign ‘‘cellular’’ form (PrP) includes a flexibly disordered 100-residue tail linked to the N-terminal end of a globular domain (Figs. 3 and 4 [3]. Considering that the mechanism of transformation of PrP into the aggregated, disease-related form of mammalian prion proteins is still subject to speculation, the observation of this flexible tail has been highly intriguing.

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.

Modification of bovine serum albumin structure following reaction with 4,5(E)-epoxy-2(E)-heptenal.

Bovine serum albumin (BSA) was incubated for different periods of time and in the presence of several concentrations of 4, 5(E)-epoxy-2(E)-heptenal, at pH 7.4 and 37 degrees C, in an effort to analyze the changes produced in its structure as a consequence of its reaction with this product of lipid oxidation. The epoxyalkenal modified the primary structure of BSA as determined by lysine losses and formation of oxidative stress product epsilon-N-pyrrolylnorleucine (Pnl), which depended on the concentration of the aldehyde and the incubation time. These changes also modified secondary and tertiary structures of the protein, which were determined by studying protein denaturation and polymerization. In addition, all these modifications were parallel to the development of color and fluorescence, which were produced as a consequence of the formation and polymerization of pyrrole amino acid residues. The above results indicated that epoxyalkenals modify the protein structure and develop color and fluorescence. A failure in the degradation of these modified proteins might induce their accumulation and, thus, participation in lipofuscin or age pigments formation.

Protein Denaturation in Foam: II. Surface Activity and Conformational Change

As part of a study of protein denaturation in foam we have measured the surface tension and the changes in protein structure occurring at the interface for lysozyme, pepsin, BSA, YADH, IgG, and catalase. The apparent CMC values were found to be dependent on the size and rigidity of the molecule. The variability of protein damage at a gas–liquid interface in foam was assessed using these proteins. The foams were produced under controlled conditions in a bubble column and were found to induce conformational changes in the protein molecules, but no fragmentation or disassociation of subunits occurred. Tertiary structural changes were detected in all the proteins studied, with some proteins forming aggregates. For pepsin, the secondary structure was also found to be altered. Enzyme solutions were used to determine the degree of biological activity retained after foaming for proteins with different structural characteristics. The more rigid proteins were found to display a low surface activity and a low degree of damage in foam. Pepsin suffered the highest rate of damage, which is thought to be a result of its inability to refold following denaturation.

Capillary zone electrophoresis with optimized temperature control for studying thermal denaturation of proteins at various pH.

Capillary electrophoresis (CE) was used to analyze the thermal denaturation of bovine beta-lactoglobulin at different pH. This model protein exhibits complex pH- and temperature association/dissociation dependence balances in its quaternary structure. The study was possible after modification and improvement of a capillary electrophoresis apparatus. The improvement allowed both efficient control (temperature fluctuations <0.05 degrees C) and accurate measurement of the temperature (+/- 0.1 degrees C) within the capillary cartridge. CE allowed the thermodynamic parameters of beta-lactoglobulin thermal denaturation to be estimated. The transition temperature, Tm, was determined at acidic, neutral and alkaline pH. Van't Hoff analysis was performed through direct measurement of native and unfolded protein populations in the slow-time regime. This allowed estimation of thermodynamic parameters (deltaH, deltaS, deltaCp). Finally, the stability curve, i.e., the temperature dependence of the free energy change (deltaG) of protein unfolding was drawn. The accuracy of the parameters values compares with parameters obtained by calorimetric measurements. The available parameters and the requirement of minute amount of protein sample are of potential interest in the field of protein engineering and biological pharmaceuticals. Accordingly, CE can be proposed as a convenient tool to study protein stability and denaturation processes.

Proteins in Vacuo. Denaturing of Disulfide-Intact and Disulfide-Broken Lysozyme Probed by Molecular Dynamics Simulations

Proteins in vacuo are the subject of a number of experimental techniques where unfolding has been shown to be an important feature. In this paper we report on a detailed structural study of protein denaturation modeled in molecular dynamics (MD) simulations of disulfide-intact (DI) and disulfide-reduced (DR) lysozyme (LYZ) molecules at 293 K in vacuo with the GROMOS force field. The trajectories were carried out over at least 1.0 ns in the absence of water molecules, starting from an X-ray structure. A repulsive centrifugal potential was generated for both DI- and DR-LYZ, inducing large conformational transitions. Denaturation followed a pathway eliciting the existence of two well-defined subdomains involving the alpha helixes (designated here as α1 and α2) while the beta sheet appeared to comprise a full domain (β). The domain unfolding differed markedly for DI- and DR-LYZ. The unusual structures found in this type of simulation for DI-LYZ were compared with related unfolding simulations in water and wer...

A model-independent, nonlinear extrapolation procedure for the characterization of protein folding energetics from solvent-denaturation data.

We have characterized the guanidine-induced denaturation of hen egg white lysozyme within the 30-75 degrees C temperature range on the basis of equilibrium fluorescence measurements, unfolding assays, kinetic fluorescence measurements, and differential scanning calorimetry. Analysis of the guanidine denaturation profiles according to the linear extrapolation method yields values for the denaturation Gibbs energy which are about 15 kJ/mol lower than those derived from differential scanning calorimetry. Our results strongly suggest that this discrepancy is not due to deviations from the two-state denaturation mechanism. We propose a new method for the determination of denaturation Gibbs energies from solvent-denaturation data (the constant-delta G extrapolation procedure). It employs several solvent-denaturation profiles (obtained at different temperatures) to generate the protein stability curve at zero denaturant concentration within the -8 to 8 kJ/mol delta G range. The method is model-independent and provides a practical, nonlinear alternative to the commonly employed linear extrapolation procedure. The application of the constant-delta G method to our data suggests that the guanidine-concentration dependence of the denaturation Gibbs energy is approximately linear over an extended concentration range but, also, that strong deviations from linearity may occur at low guanidine concentrations. We tentatively attribute these deviations to the abrupt change of the contribution to protein stability that arises from pairwise charge-charge electrostatic interactions. This contribution may be positive, negative, or close to zero, depending on the pH value and the charge distribution on the native protein surface [Yang, A.-S., & Honig, B. (1993) J. Mol. Biol. 231, 459-474], which may help to explain why disparate effects have been found when studying protein denaturation at low guanidine concentrations. Kinetic m values for lysozyme denaturation depend on temperature, in a manner which appears consistent with Hammond behavior.

Differential scanning calorimetry: A useful tool for studying protein denaturation

Power-compensated differential scanning calorimetry is applied to the determination of the thermodynamic parameters of protein denaturation. The heating of relatively high mass samples (≈40 mg) is programmed at relatively high scan rates (10°C min −1). For these dynamic thermal measurements, attention is paid to the problems of sample thermal lag and to the construction of the baseline under the calorimetric curve. Simplified mathematical models are applied to the case of the denaturation transition of β-lactoglobulin and α-lactalbumin dispersed in distilled water at relatively high concentration (≈5% w/w). The effects of Na + and Ca 2+ addition on the conformational stability of the proteins upon thermal treatment are compared at pH 3.5 and 7, where these two major whey proteins display different thermal behaviours.

States and functions of tyrosine residues in Escherichia coli asparaginase II.

The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wild-type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active-site residue Y25 has a normal pKa while the C-terminal Y326 is unusually acidic. 1H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme.

Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants.

Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies.

A method to study protein denaturation by measurements of apparent molar volumes

Protein denaturation was studied by measurements of apparent molar volumes of bovine serum albumin (BSA)-water-urea systems at 25°C. The viscosity measurements carried out on aqueous BSA solutions at various fixed concentrations as a function of temperature have shown that the denaturation process can best be followed at 60gkg −1 BSA solution. The apparent molar volumes of urea in water and in 60gkg −1 BSA solution were measured at 25°C and the differences between the two were interpreted in terms of structural changes taking place during denaturation of BSA by urea. A simple method was proposed to calculate the volume change per mole of protein upon denaturation. The sample calculation for BSA has shown that there is a volume contraction of 1854 cm 3 per mol BSA when the urea concentration reaches 13 M.

Protein characterization by DSC

Proteins show a typical DSC fingerprint curve. DSC investigation may be used to study protein denaturation, and even for quality control.

Kinetics of thermal inactivation of avidin

Thermal destruction of the biotin-binding activity of avidin was estimated by thermal destruction time (TDT) experiments between 73·3°C and 125·6°C. Decimal reduction time at 121·1°C was 24·5 min while the response to temperature change was described by a z value of 33 C°. Heat stability of the biotin-binding site of avidin was shown to be much greater than previously inferred from DSC protein denaturation studies of avidin.

Conformational changes in melittin upon complexation with an anionic melittin analog

Melittin and its Glu-(7,21,22,23,24) analog upon mixing in equimolar concentrations form a hybrid oligomer with significant helical structure, in conditions in which each peptide separately adopts a largely disordered structure. The hybrid exhibits both cold- and heat-induced denaturations similar to the phenomena exhibited by proteins. The hybrid also retains significant residual structure at higher temperature, similar to the ‘molten globular state’ that has been suggested for proteins. Melittin, at concentrations in which it forms helical tetramers, also exhibits these phenomena and may be used as a model for protein-denaturation studies.